U.S. patent number 7,658,994 [Application Number 10/896,392] was granted by the patent office on 2010-02-09 for substrates and compounds bonded thereto.
This patent grant is currently assigned to 3M Innovative Properties Company. Invention is credited to Brinda B. Lakshmi.
United States Patent |
7,658,994 |
Lakshmi |
February 9, 2010 |
**Please see images for:
( Certificate of Correction ) ** |
Substrates and compounds bonded thereto
Abstract
Articles and methods for the use of such articles are described
for use in immobilizing nucleophile-containing materials. In one
aspect, the invention provides an article comprising: a substrate
having a first surface and a second surface; and a phosphonitrilic
tethering group affixed to the first surface of the substrate, the
phosphonitrilic tethering group comprising a reaction product of a
complementary functional group on the first surface of the
substrate with a phosphonitrilic tethering compound. A method of
immobilizing a nucleophile-containing material to a substrate is
also described, the method comprising: providing a phosphonitrilic
tethering compound; providing a substrate having a complementary
functional group capable of reacting with phosphonitrilic tethering
compound; preparing a substrate-attached phosphonitrilic tethering
group by reacting the phosphonitrilic tethering compound with the
complementary functional group on the substrate resulting in an
ionic bond, covalent bond, or combinations thereof; and reacting
the substrate-attached phosphonitrilic tethering group with a
nucleophile-containing material to immobilize the
nucleophile-containing material.
Inventors: |
Lakshmi; Brinda B. (Woodbury,
MN) |
Assignee: |
3M Innovative Properties
Company (St. Paul, MN)
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Family
ID: |
34704351 |
Appl.
No.: |
10/896,392 |
Filed: |
July 22, 2004 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20050142296 A1 |
Jun 30, 2005 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60533178 |
Dec 30, 2003 |
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Current U.S.
Class: |
428/343;
428/355R; 428/344; 428/304.4 |
Current CPC
Class: |
G01N
33/54353 (20130101); Y10T 428/2852 (20150115); Y10T
428/2804 (20150115); Y10T 428/28 (20150115); Y10T
428/249953 (20150401) |
Current International
Class: |
B32B
7/12 (20060101); B32B 15/04 (20060101); B32B
3/26 (20060101) |
Field of
Search: |
;428/343,344,355R,304.4 |
References Cited
[Referenced By]
U.S. Patent Documents
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5043600 |
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Feb 1993 |
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JP |
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WO 95/19184 |
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Jul 1995 |
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WO |
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WO 95/32736 |
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Dec 1995 |
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WO |
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WO 98/14610 |
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Apr 1998 |
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WO |
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WO 01/66244 |
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Sep 2001 |
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WO |
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WO01/66820 |
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Sep 2001 |
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WO |
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WO03/050237 |
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Jun 2003 |
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WO |
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WO 03/091304 |
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Nov 2003 |
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WO |
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WO 03/093785 |
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Nov 2003 |
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WO |
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Other References
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Pendant Polymers: Synthesis, Characterization, and Phosphate Ester
Hydrolysis Using a Cu(II)-Metalated Cross-Linked Polymeric
Catalyst, Inorganic Chemistry Article, vol. 41, No. 20, 2002, pp.
5162-5173. cited by other .
K.C. Das; Y. Lin; B. Weinstein, Experientia, "A New Peptide
Coupling Agent--Phosphonitrilic Chloride," Dec. 15, 1969, pp.
1238-1239. cited by other .
H.-D. Hunger, CH. Coutelle, G. Behrendt, CHR. Flachmeier, A.
Rosenthal, A. Speer, H. Breter, R. Szargan, P. Franke, J. Stahl,
N.V. Cuong, and G. Barchend, Analytical Biochemistry, "CCA Paper: A
New Two-Dimensional Cyanuric Chloride-Activated Matrix for
Universal Application in Molecular Biology," Nov. 13, 1985, pp.
286-299. cited by other .
H. Allcock and S. Kwon, Macromolecules 1986, "Covalent Linkage of
Proteins to Surface-Modified Poly(organophosphazenes):
Immobilization of Glucose-6-Phosphate Dehydrogenase and Trypsin,"
Jan. 7, 1986, pp. 1502-1508. cited by other .
H.-D. Hunger, A. Speer, CHR. Flachmeier, R. Hanke, G. Behrendt, and
CH. Coutelle, Analytical Biochemistry, "Use of Cyanuric
Chloride-Activated Paper for Detection of Subpicogram Quantities of
Specific DNA Sequences and Its Application to Linked Restriction
Fragment Length Polymorphism Analysis in a Duchenne Muscular
Dystrophy Affected Family," Oct. 6, 1986, pp. 45-55. cited by other
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J. Van Ness, S. Kalbfleisch, C. Petrie, M. Reed, J. Tabone and N.
Vermeulen, Nucleic Acids Research, vol. 19, No. 12, "A Versatile
Solid Support System for Oligodeoxynucleotide Probe-Based
Hybridization Assays," Mar. 5, 1991, pp. 3345-3350. cited by other
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J. Mark, H. Allcock, R. West, Inorganic Polymers, Polymeric Science
& English Series, Chapter 3, "Polyphosphazenes," 1992, pp.
60-140. cited by other .
H. Allcock, C. Nelson, & W. Coggio, Chemistry of Materials,
1994, 6. "Photoinitiated Graft Poly(organophosphazenes):
Functionalized Immobilization Substrates for the Binding of Amines,
Proteins, and Metals," Jul. 15, 1993, pp. 516-524. cited by other
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H. Baek, Y. Cho, C. Lee, & Y. Sohn, Anti-Cancer Drugs 2000,
vol. 11, "Synthesis and Antitumor Activity of
Cyclotriphosphazene-(diamine)platinum(II) Conjugates," pp. 715-725.
cited by other .
Scham D. et al., "Spatially Addressed Synthesis of Amino- and
Amino-Oxy-Substituted 1,3,5-Triazine Arrays on Polymeric
Membranes", Journal of Combinatorial Chemistry American Chemical
Society, vol. 2, Jun. 2000, pp. 361-369. cited by other .
Stankova M. et al., "Library Generation Through Successive
Substitution of Trichlorotriazine", Molecular Diversity, vol. 2,
No. 1/2, 1996, pp. 75-80. cited by other.
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Primary Examiner: Chang; Victor S
Parent Case Text
This application claims the benefit of U.S. Provisional Application
No. 60/533,178, filed Dec. 30, 2003.
Claims
What is claimed is:
1. An article comprising: a substrate having a first surface and a
second surface, the first surface comprising a functional group; a
phosphonitrilic tethering group ionically or covalently bonded to
the first surface of the substrate, the phosphonitrilic tethering
group comprising a reaction product of the functional group on the
first surface of the substrate with a phosphonitrilic moiety;
wherein the substrate comprises metal or metal oxide.
2. The article according to claim 1, wherein the substrate
comprises metal or metal oxide selected from the group consisting
of gold, silver, titanium, platinum, palladium, aluminum, copper,
chromium, iron, cobalt, nickel, zinc, stainless steel, indium tin
oxide, and combinations of two or more of the foregoing.
3. The article according to claim 1, wherein the substrate further
comprises a support layer supporting the metal or metal oxide.
4. The article according to claim 3, wherein the support layer
comprises a polymer.
5. The article according to claim 1 wherein the phosphonitrilic
tethering compound comprises a structure according to Formula I
##STR00006## wherein each X may be the same or different and
comprise reactive groups susceptible to nucleophilic attack to bond
with a nucleophile-containing material.
6. The article according to claim 5 wherein each X is chlorine.
7. The article according to claim 1 further comprising a
monofunctional, difunctional, or multifunctional moiety affixed to
the phosphonitrilic tethering group.
Description
FIELD OF THE INVENTION
The invention relates to articles comprising a substrate having a
tethering group affixed to the substrate and to methods for
immobilizing a nucleophile-containing material to the
substrate.
BACKGROUND OF THE INVENTION
The covalent attachment of biologically active molecules to the
surface of a substrate can be useful in a variety of applications
such as in diagnostic devices, affinity separations, high
throughput DNA sequencing applications, the clean-up of polymerase
chain reactions (PCR), and the like. Immobilized biological amines,
for example, can be used for the medical diagnosis of a disease or
genetic defect or for detection of various biomolecules.
The modification of solid supports (e.g. particulate chromatography
supports) by introduction of reactive functional groups for the
immobilization of any of a variety of ligands is known. The
attachment of a nucleophile (e.g., NH2, SH, OH, etc.) to a
substrate may be achieved through the use of tethering compounds. A
tethering compound has at least two reactive functional groups
separated by a linking group. One of the functional groups provides
a means for anchoring the tethering compound to a substrate or
support by reacting with a complementary functional group on the
surface of the substrate. A second reactive functional group can be
selected to react with the nucleophile-containing material.
Supports containing hydroxyl groups (e.g. cellulose, cross-linked
dextrans, wool, and polyvinyl alcohol) may be treated with cyanuric
chloride (trichlorotriazine) for the attachment of enzymes,
antigens, and antibodies. Hydroxyl-containing supports such as
Sepharose may be reacted with trichlorotriazine (TCT) which may
then bind one or more nucleophiles. Solid nylon beads derivatized
with cyanuric chloride have been used for oligonucleotide based
hybridization assays. TCT coated paper and nylon membranes have
also demonstrated utility in transfer hybridization experiments of
DNA, RNA, and proteins.
Known tethering compounds are typically highly reactive with
nucleophile-containing materials including biological materials.
But, the reaction of the tethering compounds to
nucleophile-containing materials may compete with other reactions,
such as the hydrolysis of the tethering compound, when reactions
with nucleophiles are conducted in aqueous solutions. Hydrolysis
can result in incomplete or inefficient immobilization of the
nucleophile-containing materials on a substrate.
There is a need for improved immobilization substrates and for
tethering compounds compatible with such substrates. Accordingly,
it is desired to provide supports and tethering compounds that are
useful for ligand immobilization in any of a variety of
applications.
SUMMARY OF THE DISCLOSURE
The present invention provides articles and methods for the use of
such articles in immobilizing nucleophile-containing materials such
as amine-containing analyte, amino acid, peptide, DNA, RNA,
protein, enzyme, organelle, immunoglobulin, and fragments and
combinations of two or more of the foregoing. The
nucleophile-containing material may comprise an amine-containing
material such as, for example, an antigen (including an antigen
bound to an antibody), an immunoglobulin or the like. In some
embodiments, the amine-containing material may be further bound to
a bacterium such as Staphylococcus aureus.
In one aspect, the invention provides an article comprising: a
substrate having a first surface and a second surface; a
phosphonitrilic tethering group attached to the first surface of
the substrate, the phosphonitrilic tethering group comprising a
reaction product of a functional group on the first surface of the
substrate with a phosphonitrilic tethering compound.
In some embodiments, the phosphonitrilic tethering compound
comprises a structure according to Formula I
##STR00001## Wherein each X may be the same or different and
comprise reactive groups susceptible to nucleophilic attack to bond
with a nucleophile-containing material.
In another aspect, the invention provides a method of immobilizing
a nucleophile-containing material to a substrate, the method
comprising:
Providing a phosphonitrilic tethering compound;
Providing a substrate having a functional group capable of reacting
with the phosphonitrilic tethering compound;
Preparing a substrate-attached phosphonitrilic tethering group by
reacting the phosphonitrilic tethering compound with the functional
group on the substrate resulting in an ionic bond, covalent bond,
or combinations thereof; and
Reacting the substrate-attached phosphonitrilic tethering group
with a nucleophile-containing material to immobilize the
nucleophile-containing material.
Certain terms used in the description of the invention will be
understood as having the following meanings:
As used herein, the terms "a", "an", and "the" are used
interchangeably with "at least one" to mean one or more of the
elements being described.
As used herein, the term "acyl" refers to a monovalent group of
formula --(CO)R where R is an alkyl group and where (CO) used
herein indicates that the carbon is attached to the oxygen with a
double bond.
As used herein, the term "acyloxy" refers to a monovalent group of
formula --O(CO)R where R is an alkyl group.
As used herein, the term "acyloxysilyl" refers to a monovalent
group having an acyloxy group attached to a Si (i.e., Si--O(CO)R
where R is an alkyl). For example, an acyloxysilyl can have a
formula --Si[O(CO)R].sub.3-nL.sub.n where n is an integer of 0 to 2
and L is a halogen or alkoxy. Specific examples include
--Si[O(CO)CH.sub.3].sub.3, --Si[O(CO)CH.sub.3].sub.2Cl, or
--Si[O(CO)CH.sub.3]Cl.sub.2.
As used herein, the term "alkoxy" refers to a monovalent group of
formula --OR where R is an alkyl group.
As used herein, the term "alkoxycarbonyl" refers to a monovalent
group of formula --(CO)OR where R is an alkyl group.
As used herein, the term "alkoxysilyl" refers to a group having an
alkoxy group attached to a Si (i.e., Si--OR where R is an alkyl).
For example, an alkoxysilyl can have a formula
--Si(OR).sub.3-n(L.sup.a).sub.n where n is an integer of 0 to 2 and
L.sup.a is a halogen or acyloxy. Specific examples include
--Si(OCH.sub.3).sub.3, --Si(OCH.sub.3).sub.2Cl, or
--Si(OCH.sub.3)Cl.sub.2.
As used herein, the term "alkyl" refers to a monovalent radical of
an alkane and includes groups that are linear, branched, cyclic, or
combinations thereof. The alkyl group typically has 1 to 30 carbon
atoms. In some embodiments, the alkyl group contains 1 to 20 carbon
atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon
atoms. Examples of alkyl groups include, but are not limited to,
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl,
n-pentyl, n-hexyl, cyclohexyl, n-heptyl, n-octyl, and
ethylhexyl.
As used herein, the term "alkyl disulfide" refers to a monovalent
group of formula --SSR where R is an alkyl group.
As used herein, the term "alkylene" refers to a divalent radical of
an alkane. The alkylene can be straight-chained, branched, cyclic,
or combinations thereof. The alkylene typically has 1 to 200 carbon
atoms. In some embodiments, the alkylene contains 1 to 100, 1 to
80, 1 to 50, 1 to 30, 1 to 20, 1 to 10, or 1 to 4 carbon atoms. The
radical centers of the alkylene can be on the same carbon atom
(i.e., an alkylidene) or on different carbon atoms.
As used herein, "aminosilane" refers to refers to a group having an
amine group attached to a Si. For example, an aminosilane can have
a formula --Si(OR.sup.1).sub.3-n[(R.sup.2)NH.sub.2].sub.n where n
is an integer of 0 to 2 and R.sup.1 is an alkyl having a carbon
chain length less than 5, R.sup.2 is another alkyl group having a
carbon chain length of at least 2. Specific examples include
3-aminopropyl triethoxysilane, 3-amino trimethoxy silane.
As used herein, the term "aralkyl" refers to a monovalent radical
of the compound R--Ar where Ar is an aromatic carbocyclic group and
R is an alkyl group.
As used herein, the term "aralkylene" refers to a divalent radical
of formula --R--Ar-- where Ar is an arylene group and R is an
alkylene group.
As used herein, the term "aryl" refers to a monovalent aromatic
carbocyclic radical. The aryl can have one aromatic ring or can
include up to 5 carbocyclic ring structures that are connected to
or fused to the aromatic ring. The other ring structures can be
aromatic, non-aromatic, or combinations thereof. Examples of aryl
groups include, but are not limited to, phenyl, biphenyl,
terphenyl, anthryl, naphthyl, acenaphthyl, anthraquinonyl,
phenanthryl, anthracenyl, pyrenyl, perylenyl, and fluorenyl.
As used herein, the term "arylene" refers to a divalent radical of
a carbocyclic aromatic compound having one to 5 rings that are
connected, fused, or combinations thereof. In some embodiments, the
arylene group has up to 5 rings, up to 4 rings, up to 3 rings, up
to 2 rings, or one aromatic ring. For example, the arylene group
can be phenylene.
As used herein, the term "azido" refers to a group of formula
--N.sub.3.
As used herein, the term "aziridinyl" refers to a cyclic monovalent
radical of aziridine having the formula
##STR00002## where R.sup.d is hydrogen or alkyl.
As used herein, the term "benzotriazolyl" refers to a monovalent
group having a benzene group fused to a triazolyl group. The
formula for a benzotriazolyl group is C.sub.6H.sub.4N.sub.3--.
As used herein, the term "carbonyl" refers to a divalent group of
formula --(CO)--.
As used herein, the term "carbonylimino" refers to a divalent group
of the formula --(CO)NR.sup.4-- where R.sup.4 is hydrogen, alkyl,
or aryl.
As used herein, the term "carbonyloxy" refers to a divalent group
of formula --(CO)O--.
As used herein, the term "carbonyloxycarbonyl" refers to a divalent
group of formula --(CO)O(CO)--. Such a group is part of an
anhydride compound.
As used herein, the term "carbonylthio" refers to a divalent group
of formula --(CO)S--.
As used herein, the term "carboxy" refers to a monovalent group of
formula --(CO)OH.
As used herein, the term "chloroalkyl" refers to an alkyl having at
least one hydrogen atom replaced with a chlorine atom.
As used herein, the term "cyano" refers to a group of formula
--CN.
As used herein, the term "disulfide" refers to a divalent group of
formula --S--S--.
As used herein, the term "ethylenically unsaturated" refers to a
monovalent group having a carbon-carbon double bond of formula
--CY.dbd.CH.sub.2 where Y is hydrogen, alkyl, or aryl.
As used herein, the term "fluoroalkyl" refers to an alkyl having at
least one hydrogen atom replaced with a fluorine atom.
As used herein, the term "haloalkyl" refers to an alkyl having at
least one hydrogen atom replaced with a halogen selected from F,
Cl, Br, or I. Perfluoroalkyl groups are a subset of haloalkyl
groups.
As used herein, the term "halocarbonyloxy" refers to a monovalent
group of formula --O(CO)X where X is a halogen atom selected from
F, Cl, Br, or I.
As used herein, the term "halocarbonyl" refers to a monovalent
group of formula --(CO)X where X is a halogen atom selected from F,
Cl, Br, or I.
As used herein, the term "halosilyl" refers to a group having a Si
attached to a halogen (i.e., Si--X where X is a halogen). For
example, the halosilyl group can be of formula
--SiX.sub.3-n(L.sup.b).sub.n where n is an integer of 0 to 2 and
L.sup.b is selected from an alkoxy, or acyloxy. Some specific
examples include the groups --SiCl.sub.3, --SiCl.sub.2OCH.sub.3,
and --SiCl(OCH.sub.3).sub.2.
As used herein, the term "heteroalkylene" refers to a divalent
alkylene having one or more carbon atoms replaced with a sulfur,
oxygen, or NR.sup.d where R.sup.d is hydrogen or alkyl. The
heteroalkylene can be linear, branched, cyclic, or combinations
thereof and can include up to 400 carbon atoms and up to 30
heteroatoms. In some embodiments, the heteroalkylene includes up to
300 carbon atoms, up to 200 carbon atoms, up to 100 carbon atoms,
up to 50 carbon atoms, up to 30 carbon atoms, up to 20 carbon
atoms, or up to 10 carbon atoms.
As used herein, the term "hydroxy" refers to a group of formula
--OH.
As used herein, the term "isocyanato" refers to a group of formula
--NCO.
As used herein, the term "mercapto" refers to a group of formula
--SH.
As used herein, "nucleophile" or "nucleophile-containing material"
refers to moieties with reactive oxygen, sulfur and/or nitrogen
containing groups such as substituted amino groups. Examples of
nucleophile-containing materials include those with moieties such
as amino, alkyl or aryl substituted amino, alkylamino, arylamino,
oxyalkyl, oxyaryl, thioalkyl, and thioaryl groups, residues of
dyestuffs containing amino groups such as nitro-dyestuffs,
azo-dystuffs, including thiazole dystuffs, acridine-, oxyazine-,
thiazine- and azine dyestuffs, indigoids, aminoanthraquinones,
aromatic diamines, aminophenols, aminonaphthols and N and O-acidyl
or alkyl, aralkyl or aryl derivatives of these, nitramines,
thiophenols, or amino mercaptans. Exemplary nucleophile-containing
material include the following moieties: OCH2 COOH; NHCH.sub.2COOH;
SCH.sub.2COOH; NHC.sub.2H.sub.4SO.sub.3H;
OC.sub.4H.sub.8N(C.sub.2H.sub.5).sub.3; NHC.sub.6H.sub.4SO.sub.3H;
OC.sub.6H.sub.4COOH; SC.sub.6H.sub.4COOH; NHC.sub.2H.sub.4OH;
OC.sub.2H.sub.4OH; and
NHC.sub.3H.sub.6NH(C.sub.2H.sub.4OH).sub.2.
As used herein, the term "oxy" refers to a divalent group of
formula --O--.
As used herein, the term "perfluoroalkyl" refers to an alkyl group
in which all of the hydrogen atoms are replaced with fluorine
atoms. Perfluoroalkyl groups are a subset of fluoroalkyl
groups.
As used herein, the term "phosphato" refers to a monovalent group
of formula --OPO.sub.3H.sub.2.
As used herein, "phosphonitrilic moiety" or "phosphonitrilic group"
refers to structures of the following general formula:
##STR00003##
As used herein, "phosphonitrilic tethering compound" or
"phosphonitrilic tethering group" refer to tethering compounds or
tethering groups having at least one phosphonitrilic moiety or
group.
As used herein, the term "phosphono" refers to a monovalent group
of formula --PO.sub.3H.sub.2.
As used herein, the term "phosphoramido" refers to a monovalent
group of formula --NHPO.sub.3H.sub.2.
As used herein, the term "primary aromatic amino" refers to a
monovalent group of formula --ArNH.sub.2 where Ar is an aryl
group.
As used herein, the term "secondary aromatic amino" refers to a
monovalent group of formula --ArNR.sup.hH where Ar is an aryl group
and R.sup.h is an alkyl or aryl.
As used herein, the term "tertiary amino" refers to a group of
formula --NR.sub.2 where R is an alkyl.
As used herein, the term "tethering compound" refers to a compound
that has at least two reactive groups. One of the reactive groups
(i.e., the substrate-reactive functional group) can react with a
complementary functional group on the surface of a substrate to
form a tethering group. Another reactive group can react either
with a nucleophile-containing material, or another tethering
compound (or a derivative or oligomer thereof) or another moiety
capable of bonding with a nucleophile-containing material. Reaction
of two reactive groups of the tethering compound results in the
formation of a connector group between the substrate and a
nucleophile-containing material such as an amine-containing
material that is immobilized on the substrate.
As used herein, the term "tethering group" refers to a group
attached to a substrate that results from the reaction of a
tethering compound with a complementary functional group on the
surface of the substrate with a tethering compound.
The foregoing summary is not intended to be inclusive of all
possible embodiments of the invention. Those skilled in the art
will more fully appreciate the features and advantages of the
invention upon consideration of the remainder of the disclosure
including the Detailed Description of the Preferred Embodiment, the
various Examples and the appended claims.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention provides articles and methods for
immobilizing nucleophile-containing materials to a substrate.
Phosphonitrilic compounds having reactive functional groups are
described for use as tethering compounds between a substrate and at
least one nucleophile-containing material. Tethering compounds
useful in the invention comprise reactive groups susceptible to
nucleophilic attack. At least one of the reactive functional groups
on the tethering compound provides a means of attachment of the
tethering compound to a surface of a substrate. The remaining
functional groups can each be reacted with a nucleophile-containing
material, such as amine functional proteins, enzymes, other
biomolecules or the like. Additionally, the functional groups can
be reacted with nucleophile-containing groups or can provide
additional links to other moieties such as other similar tethering
compounds or other reactive moieties which may be simple or complex
in their structures (e.g., branched, straight chain, etc.) and
typically including additional reactive groups that are also
capable of bonding with nucleophile-containing materials.
In embodiments of the invention, tethering compounds for bonding
biological molecules to the surface of a substrate comprise
phosphonitrilic groups, and may be of the general composition of
Formula I:
##STR00004## Wherein
Each X may be the same or different and comprise reactive groups
susceptible to nucleophilic attack to bond with a
nucleophile-containing material. Typically, X includes a halogen
and most typically, X is chlorine.
Phosphonitrilic tethering compounds useful in the present invention
include phosphonitrilic chloride trimer ("PNC") wherein each X in
Formula I is chlorine. In tethering the PNC to a substrate, at
least one of the chlorines is reacted with a moiety on the surface
of a substrate to bond the PNC moiety to the substrate. When the
PNC moiety is bonded to the substrate, the phosphonitrilic
tethering compound includes additional reactive groups, each
capable of reacting with a nucleophile-containing material, such as
a biologically active material, to tether the biologically active
material to the substrate through the phosphonitrilic moiety.
In some embodiments of the invention, the phosphonitrilic tethering
groups may be derived solely from PNC molecules. In some
embodiments, the phosphonitrilic tethering groups may be derived
from compounds considered to be oligomers or derivatives of PNC.
Referring to Formula I, tethering groups derived solely from PNC
are those compounds of Formula I wherein each X is chlorine.
Oligomers of phosphonitrilic trimer suitable for use in the present
invention include compounds of Formula I wherein at least one of
the X groups is chlorine.
Derivatives of PNC suitable for inclusion in the phosphonitrilic
tethering groups of the present invention include compounds of
Formula I wherein at least one X is substituted with a moiety that
may include monofunctional groups, difunctional groups or other
multifunctional groups wherein the functional groups are typically
nucleophiles. Such functional groups may be organic moieties that
may be, in whole or in part, aliphatic (straight chain or branched
chain) or aromatic. In some embodiments, the monofunctional,
difunctional and/or multifunctional moieties may be bonded to a
phosphonitrilic moiety prior to the attachment of the
phosphonitrilic moiety to the substrate. In some embodiments, the
monofunctional, difunctional and/or multifunctional moieties may be
bonded to a phosphonitrilic moiety after the phosphonitrilic moiety
has already been attached (e.g., bonded) to a substrate.
In embodiments where the phosphonitrilic moiety is derived from
PNC, the reaction of the chlorines (X of Formula I are all
chlorine) is typically sequential and the reactivity of each
chlorine depends on the number of chlorines remaining on the PNC
molecule, the nature of the moiety being reacted with the PNC
(e.g., nucleophilicity, steric hindrance) and the reaction
conditions (temperature, presence of water, stoichiometry or
reactants, etc.). Where one of group X, for example, of Formula I
is reacted with a moiety on the surface of a substrate to bond the
phosphonitrilic moiety to the substrate, the remaining unreacted X
groups remain capable of reacting with nucleophile-containing
materials including monofunctional, difunctional and/or
multifunctional moieties.
Monofunctional groups include moieties with a reactive group (e.g.,
nucleophiles) capable of reacting with one of the X groups of the
compounds of Formula I but generally do not include additional
reactive groups. In some embodiments, monofunctional groups may
comprise groups having one or more desired properties that are
needed or desired in the substrates or the tethering groups of the
present invention. Suitable monofunctional groups include groups
that render the reaction product hydrophilic or hydrophobic, groups
that enhance solubility in certain solvents, groups that enhance
molecular interactions, and the like. Examples include
monofunctional organic alcohols, amines and mercaptans.
Difunctional groups may be linking groups in that they include a
first reactive group that can react with a phosphonitrilic moiety
and a second reactive group that can also react with the
phosphonitrilic moiety or it can react with another compound or
moiety including a second compound of Formula I such as PNC, for
example. In some embodiments difunctional groups comprise linking
groups that can link phosphonitrilic moieties to one another to
form a tethering group comprised of at least two phosphonitrilic
moieties connected to one another through the difunctional linking
group. In such a configuration, the phosphonitrilic moieties will
include unreacted groups (e.g., unreacted X groups according to
Formula I) capable of bonding with other nucleophile-containing
materials such biologically active molecules, for example. In some
embodiments, the unreacted groups may comprise chlorines on one,
two or more phosphonitrilic moieties tethered or linked together
through one or more difunctional linking groups. In some
embodiments, the difunctional groups can react with two reactive
groups on the same phosphonitrilic moiety (two X groups of Formula
I).
Suitable difunctional moieties include compounds having two
reactive groups such as two nucleophilic groups. Some specific
difunctional moieties include, for example, 4,7,10-trioxa-1,
13-tridecane diamine, 1,6-hexanediamine, methyl-oxirane,
p-phenylenediamine, 2-aminoethanol, 4,4-thiobisbenzenethiol,
dimethyl-1,6-hexanediamine. Other difunctional moieties will be
known to those of skill in the art, and the invention is not to be
limited in any respect to the foregoing specific moieties.
Multifunctional moieties may also comprise linking groups in that
they include a first reactive group that can react with a first
phosphonitrilic moiety bonded to a substrate, and second, third and
possibly other additional reactive groups that can react with the
same phosphonitrilic moiety or other compounds or moieties
including other phosphonitrilic moieties or compounds of Formula I
(e.g., TCT). In some embodiments multifunctional groups include
linking groups that can link two or more phosphonitrilic moieties
to one another to form a branched tethering group comprised of two
or more phosphonitrilic moieties linked together through the
multifunctional linking group. In such a configuration, the
phosphonitrilic moieties will include unreacted groups (e.g.,
unreacted X groups according to Formula I) capable of bonding with
other nucleophile-containing materials such as one or more
biologically active molecules, for example. In some embodiments,
the unreacted groups may comprise chlorines on one, two or more
phosphonitrilic moieties tethered or linked together through one or
more multifunctional linking groups. In some embodiments, the
multifunctional linking group may react with more than one reactive
group on a first phosphonitrilic group and then may also react with
other reactive groups on other phosphonitrilic groups or other
groups.
Suitable multifunctional moieties include compounds having more
than two reactive groups (e.g., nucleophilic groups). In some
embodiments, the multifunctional moieties may be oligomeric or
polymeric moieties. Some specific multifunctional moieties include,
for example, hydrolyzed 2-ethyl-4,5-dihydro-oxazole homopolymer,
polyethylenimine (including linear and branched configurations), as
well as other moieties known to those of ordinary skill.
It will be understood that the foregoing description should not be
interpreted as limited to the specific monofunctional, difunctional
or other multifunctional groups described herein. The present
invention is intended to encompass tethering compounds and
tethering groups that include at least one phosphonitrilic
moiety.
The invention provides articles that include a phosphonitrilic
tethering group, as described herein, attached to a substrate. The
substrate-attached tethering group is the reaction product of a
complementary functional group "G" on a surface of a substrate with
at least one of the X groups of compounds of Formula I. The
substrate-attached tethering group has at least one, typically two
or more reactive groups that can react with another molecule or
materials (e.g., a nucleophile-containing material) to capture the
material and tether it to the substrate.
The substrate is a solid phase material to which the
phosphonitrilic tethering compounds can be attached. The substrate
is not soluble in a solution or solvent that might be used when
attaching a compound of Formula I to the surface of the substrate.
Typically, a phosphonitrilic tethering compound is attached only to
an outer portion of the substrate while the remaining portions of
the substrate are not modified during the process of attaching
phosphonitrilic tethering groups to the substrate. If the substrate
has groups "G" distributed throughout the substrate, typically only
those groups in the outer portion (e.g., on or near the surface)
are usually capable of reacting with an X group of the compounds
according to Formula I.
The substrates can have any useful form including, but not limited
to, thin films, sheets, membranes, filters, nonwoven or woven
fibers, hollow or solid beads, bottles, plates, tubes, rods, pipes,
or wafers. The substrates can be porous or non-porous, rigid or
flexible, transparent or opaque, clear or colored, and reflective
or non-reflective. Suitable substrate materials include, for
example, polymeric materials, glasses, ceramics, metals, metal
oxides, hydrated metal oxides, or combinations thereof.
The substrates can be a single layer or material or can have
multiple layers of one or more materials. For example, the
substrate can have one or more inner or first layers that provide
support for the outermost layer wherein the outer layer of the
substrate includes a complementary functional group capable of
reacting with the X group in compound of Formula I. In some
embodiments, a surface of an outer layer may be chemically modified
or coated with another material to provide an outer layer that
includes a complementary functional group capable of reacting with
a phosphonitrilic group including groups according to Formula
I.
Suitable polymeric materials for use as a substrate or as a portion
of a substrate include, but are not limited to, polyolefins,
polystyrenes, polyacrylates, polymethacrylates, polyacrylonitriles,
poly(vinylacetates), polyvinyl alcohols, polyvinyl chlorides,
polyoxymethylenes, polycarbonates, polyamides, polyimides,
polyurethanes, phenolics, polyamines, amino-epoxy resins,
polyesters, silicones, cellulose based polymers, polysaccharides,
or combinations thereof. In some embodiments, the polymeric
material is a copolymer prepared using a co-monomer having a
complementary functional group capable of reacting with a
phosphonitrilic group including group X in compounds according to
Formula I. For example, the co-monomer can contain a carboxy,
mercapto, hydroxy, amino, or alkoxysilyl group.
In some embodiments, suitable polymeric materials include those
resulting from thermally induced phase separation ("TIPS") which is
a phase inversion method in which an initially homogeneous polymer
solution is cast and exposed to a cooler interface (e.g., a water
bath or chilled casting wheel), and phase separation is induced in
the solution film by lowering the temperature. Suitable TIPS films
or membranes may possess a broad range of physical film properties
and microscopic pore sizes. They may be relatively rigid or
non-rigid substrates prepared from any of a variety of polymers.
TIPS membranes made according to the teachings of U.S. Pat. Nos.
4,539,256, 5,120,594, and 5,238,623 are all suitable for use in the
invention. The TIPS membranes may comprise high density
polyethylene (HDPE), polypropylene, polyvinylidenefluoride (PVDF),
polyethylene-vinyl alcohol copolymer (e.g., available under the
trade designation EVAL F101A from EVAL Company of America (EVALCA),
Houston, Tex.), for example. The membrane may comprise a
combination of materials such as a TIPS HDPE or a polypropylene
membrane coated with a hydrophilic polymer (e.g.,
polyethylene-vinyl alcohol copolymer or EVAL) or a TIPS
polypropylene support coated with a hydrophilic, strongly basic
positively-charged coating such as polydiallyldimethylammonium
chloride or a polymer incorporating quaternized
dimethylaminoethylacrylate. Another example of a suitable TIPS
membrane for use in the present invention is an HDPE membrane
commercially available from 3M Company of St. Paul, Minn. Features
of such a membrane include a pore size of about 0.09 um with a
thickness of about 0.9 mil (0.023 mm). In general, the TIPS
technology can provide a broad range of physical film properties
having pore sizes in the micro- and ultrafiltration range such as
those comprising a pore diameter within the range from about 80 nm
to about 0.5 micrometer.
Combinations of materials may be used as a solid support member and
the foregoing description is to be understood to include the
aforementioned materials alone and in combination with other
materials.
Some embodiments of the invention may utilize a multi-layered
substrate having a diamond like glass (DLG) coating applied to a
TIPS membrane or over another polymer substrate. The DLG coating
may be applied using known techniques such as by a plasma
deposition process according to that described in EP 1 266 045 B1
(David et al). In embodiments with a TIPS substrate, a DLG coating
is typically applied over the entire surface of the TIPS membrane
so that the DLG extends into the pores of the TIPS membrane. As
mentioned, other materials may be used in the manufacture of a TIPS
membrane, and a DLG coating may similarly be applied to such other
materials in order to provide a suitable substrate for use in the
present invention.
Suitable glass and ceramic substrate materials can include, for
example, sodium, silicon, aluminum, lead, boron, phosphorous,
zirconium, magnesium, calcium, arsenic; gallium, titanium, copper,
or combinations thereof. Glasses typically include various types of
silicate containing materials.
In some embodiments, the substrate includes a layer of diamond-like
glass such as is described in International Patent Application WO
01/66820 A1, the disclosure of which is incorporated herein by
reference in its entirety. The diamond-like glass is an amorphous
material that includes carbon, silicon, and one or more elements
selected from hydrogen, oxygen, fluorine, sulfur, titanium, or
copper. Some diamond-like glass materials are formed from a
tetramethylsilane precursor using a plasma process. A hydrophobic
material can be produced that is further treated in an oxygen
plasma to control the silanol concentration on the surface.
Diamond-like glass can be in the form of a thin film or in the form
of a coating on another layer or material in the substrate. In some
applications, the diamond-like glass can be in the form of a thin
film having at least 30 weight percent carbon, at least 25 weight
percent silicon, and up to 45 weight percent oxygen. Such films can
be flexible and transparent. In some embodiments, the diamond-like
glass is the outer layer of a multilayer substrate. In a specific
example, the second layer (e.g., support layer) of the substrate is
a polymeric material and the first layer is a thin film of
diamond-like glass. The tethering group is attached to the surface
of the diamond-like glass.
In some multilayer substrates, the diamond like glass is deposited
on a layer of diamond-like carbon. For example, the second layer
(e.g., support layer) is a polymeric film having a layer of
diamond-like carbon deposited on a surface. A layer of diamond-like
glass is deposited over the diamond-like carbon layer. In some
embodiments, the diamond-like carbon is a tie layer or primer layer
between a polymeric layer and a layer of diamond-like glass in a
multilayer substrate. For example, the multilayer substrate can
include a polyimide or polyester layer, a layer of diamond-like
carbon deposited on the polyimide or polyester, and a layer of
diamond-like glass deposited on the diamond-like carbon. In another
example, the multilayer substrate includes a stack of the layers
arranged in the following order: diamond-like glass, diamond-like
carbon, polyimide or polyester, diamond-like carbon, and
diamond-like glass.
Diamond-like carbon films can be prepared, for example, from
acetylene in a plasma reactor. Other methods of preparing such
films are described U.S. Pat. Nos. 5,888,594 and 5,948,166 as well
as in the article M. David et al., AlChE Journal, 37 (3), 367-376
(March 1991), the disclosures of which are incorporated herein by
reference.
Suitable metals, metal oxides, or hydrated metal oxides for
substrates can include, for example, gold, silver, platinum,
palladium, aluminum, copper, chromium, iron, cobalt, nickel, zinc,
and the like. The metal-containing material can be alloys such as
stainless steel, indium tin oxide, and the like. In some
embodiments, a metal-containing material is the top layer of a
multilayer substrate. For example, the substrate can have a
polymeric second layer and a metal containing first layer. In one
example, the second layer is a polymeric film and the first layer
is a thin film of gold. In other examples, a multilayer substrate
includes a polymeric film coated with a titanium-containing layer
and then coated with a gold-containing layer. That is, the titanium
layer can function as a tie layer or a primer layer for adhering
the layer of gold to the polymeric film.
In other examples of a multilayer substrate, a silicon support
layer is covered with a layer of chromium and then with a layer of
gold. The chromium layer can improve the adhesion of the gold layer
to the silicon layer.
The surface of the substrate typically includes a group capable of
reacting with a carboxy, halogen (e.g., chlorine), halocarbonyl,
halocarbonyloxy, cyano, hydroxy, mercapto, isocyanato, halosilyl,
alkoxysilyl, acyloxysilyl, azido, haloalkyl, tertiary amino,
primary aromatic amino, secondary aromatic amino, disulfide, alkyl
disulfide, benzotriazolyl, phosphonitrilic, phosphono,
phosphoroamido, phosphato, or ethylenically unsaturated group. That
is, the substrate includes a group "G" capable of reacting with the
group X in compounds of Formula I (i.e., the substrate includes a
complementary functional group to the group X). Substrates can
include a support material that is treated to have an outer layer
that includes a complementary functional group. The substrate can
be prepared from any solid phase material known to have groups
capable of reacting with X or which is capable of reacting with an
intermediate compound that can act as a linking group by reacting
with a moiety on the surface of the substrate and with X to link
the substrate and the phosphonitrilic group together.
A carboxy group or a halocarbonyl group can react with a substrate
having a hydroxy group to form a carbonyloxy-containing attachment
group. Examples of substrate materials having hydroxy groups
include, but are not limited to, polyvinyl alcohol, corona-treated
polyethylene, hydroxy substituted esters of polymethacrylates,
hydroxy substituted esters of polyacrylates, and a polyvinyl
alcohol coating on a support material such as glass or polymer
film.
A carboxy group or a halocarbon group can also react with a
substrate having a mercapto group to form a carbonylthio-containing
attachment group. Examples of substrate materials having a mercapto
group include, but are not limited to, mercapto substituted esters
of polyacrylates, mercapto substituted esters of polymethacrylates,
and glass treated with a mercaptoalkylsilane.
Additionally, a carboxy group or a halocarbonyl group can react
with a primary aromatic amino group, a secondary aromatic amino
group, or a secondary aliphatic amino group to form a
carbonylimino-containing attachment group. Examples of substrate
materials having aromatic primary or secondary amino groups
include, but are not limited to, polyamines, amine substituted
esters of polymethacrylate, amine substituted esters of
polyacrylate, polyethylenimines, and glass treated with an
aminoalkylsilane.
A halocarbonyloxy group can react with a substrate having a hydroxy
group to form an oxycarbonyloxy-containing attachment group.
Examples of substrate materials having hydroxy groups include, but
are not limited to, polyvinyl alcohol, corona-treated polyethylene,
hydroxy substituted esters of polymethacrylates, hydroxy
substituted esters of polyacrylates, and a polyvinyl alcohol
coating on a support material such as glass or polymer film.
A halocarbonyloxy group can also react with a substrate having a
mercapto group to form an oxycarbonylthio-containing attachment
group. Examples of substrate materials having a mercapto group
include, but are not limited to, mercapto substituted esters of
polymethacrylates, mercapto substituted esters of polyacrylates,
and glass treated with a mercaptoalkylsilane.
Additionally, a halocarbonyloxy group can react with a substrate
having a primary aromatic amino group, a secondary aromatic amino
group, or a secondary aliphatic amino group to form an
oxycarbonylimino-containing attachment group. Examples of substrate
materials having aromatic primary or secondary amino groups
include, but are not limited to, polyamines, amine substituted
esters of polymethacrylate, amine substituted esters of
polyacrylate, polyethylenimines, and glass treated with an
aminoalkylsilane.
A hydroxy group can react with a substrate having isocyanate group
to form an oxycarbonylimino-containing attachment group. Suitable
substrates having isocyanate groups include, but are not limited
to, a coating of 2-isocyanatoethylmethacrylate polymer on a support
material. Suitable support materials include glass and polymeric
materials such as polyesters, polyimides, and the like.
A hydroxy group can also react with a substrate having a carboxy,
carbonyloxycarbonyl, or halocarbonyl to form a
carbonyloxy-containing attachment group. Suitable substrates
include, but are not limited to, a coating of acrylic acid polymer
or copolymer on a support material or a coating of a methacrylic
acid polymer or copolymer on a support material. Suitable support
materials include glass and polymeric materials such as polyesters,
polyimides, and the like. Other suitable substrates include
copolymers of polyethylene with polyacrylic acid, polymethacrylic
acid, or combinations thereof.
A mercapto group can react with a substrate having isocyanate
groups. The reaction between a mercapto group and an isocyanate
group forms a thiocarbonylimino-containing attachment group.
Suitable substrates having isocyanate groups include, but are not
limited to, a coating of 2-isocyanatoethylmethacrylate copolymer on
a support material. Suitable support materials include glass and
polymeric materials such as polyesters, polyimides, and the
like.
A mercapto group can also react with a substrate having a
halocarbonyl group to form a carbonylthio-containing attachment
group. Substrates having halocarbonyl groups include, for example,
chlorocarbonyl substituted polyethylene.
A mercapto group can also react with a substrate having a
halocarbonyloxy group to form an oxycarbonlythio-containing
attachment group. Substrates having halocarbonyl groups include
chloroformyl esters of polyvinyl alcohol.
Additionally, a mercapto group can react with a substrate having an
ethylenically unsaturated group to form a thioether-containing
attachment group. Suitable substrates having an ethylenically
unsaturated group include, but are not limited to, polymers and
copolymers derived from butadiene.
A phosphonitrilic moiety such as PNC can react with
nucleophile-containing materials including glass, diamond-like
glass, metal, metal oxide and polymeric substrates with nucleophile
functionality. DLG surfaces may be treated to comprise a surface
comprising a nucleophile such as an aminosilane (e.g.,
3-aminopropyl triethoxysilane, 3-amino trimethoxy silane) that will
provide the necessary functionality to react with a PNC moiety.
Such a surface my also comprise a porous polymeric coating (e.g.,
TIPS materials described herein). Polymeric substrates can also
include, for example, ammonia grafted sintered polyethylene,
aminated polyester blown melt fiber membrane, hydroxylated
polypropylene, polyester, and polyethylene blown melt fiber
membrane, and aminomethylated styrene divinylbenzene. PNC materials
may also be reacted with metal or metal oxide materials.
An isocyanate group can react with a substrate having a hydroxy
group to form a oxycarbonylimino-containing attachment group.
Examples of substrate materials having hydroxy groups include, but
are not limited to, polyvinyl alcohol, corona-treated polyethylene,
hydroxy substituted esters of polymethacrylates or polyacrylates,
and a polyvinyl alcohol coating on glass or polymer film.
An isocyanate group can also react with a mercapto group to form a
thiocarbonylimino-containing attachment group. Examples of
substrate materials having a mercapto group include, but are not
limited to, mercapto substituted esters of polymethacrylates or
polyacrylates and glass treated with a mercaptoalkylsilane.
Additionally, an isocyanate group can react with a primary aromatic
amino group, a secondary aromatic amino group, or a secondary
aliphatic amino group to form a urea-containing attachment group.
Suitable substrates having a primary or secondary aromatic amino
group include, but are not limited to, polyamines,
polyethylenimines, and coatings of an aminoalkylsilane on a support
material such as glass or on a polymeric material such as a
polyester or polyimide.
An isocyanate group can also react with a carboxy to form an O-acyl
carbamoyl-containing attachment group. Suitable substrates having a
carboxylic acid group include, but are not limited to, a coating of
an acrylic acid polymer or copolymer or a coating of a methacrylic
acid polymer or copolymer on a glass or polymeric support.
Copolymers include, but are not limited to, copolymers that contain
polyethylene and polyacrylic acid or polymethacrylic acid. Suitable
polymeric support materials include polyesters, polyimides, and the
like.
A halosilyl group, an alkoxysilyl group, or an acyloxysilyl group
can react with a substrate having a silanol group to form a
disiloxane-containing attachment group. Suitable substrates include
those prepared from various glasses, ceramic materials, or
polymeric material. These groups can also react with various
materials having metal hydroxide groups on the surface to form a
silane-containing linkage. Suitable metals include, but are not
limited to, silver, aluminum, copper, chromium, iron, cobalt,
nickel, zinc, and the like. In some embodiments, the metal is
stainless steel or another alloy. Polymeric material can be
prepared to have silanol groups. For example, commercially
available monomers with silanol groups include
3-(trimethoxysilyl)propyl methacrylate and
3-aminoproplytrimethoxysilane available from Aldrich Chemical Co.,
Milwaukee, Wis.
An azido group can react, for example, with a substrate having
carbon-carbon triple bond to form triazolediyl-containing
attachment groups. An azido group can also react with a substrate
having nitrile groups to form a tetrazenediyl-containing attachment
group. Substrates having nitrile groups include, but are not
limited to, coatings of polyacrylonitrile on a support material
such as glass or a polymeric material. Suitable polymeric support
material includes polyesters and polyimides, for example. Other
suitable substrates having nitrile groups include acrylonitrile
polymers or copolymers and 2-cyanoacrylate polymers or
copolymers.
An azido group can also react with a strained olefinic group to
form a triazolediyl-containing attachment group. Suitable
substrates have a strained olefinic group include coatings that
have pendant norbornenyl functional groups. Suitable support
materials include, but are not limited to, glass and polymeric
materials such as polyesters and polyimides.
An aziridinyl group can react with a mercapto group to form a
aminoalkylthioether-containing attachment group. Examples of
substrate materials having a mercapto group include, but are not
limited to, mercapto substituted esters of poly methacrylates or
polyacrylates and glass treated with a mercaptoalkylsilane.
Additionally, an aziridinyl group can react with a carboxy group to
form a .beta.-aminoalkyloxycarbonyl-containing attachment group.
Suitable substrates having a carboxy include, but are not limited
to, a coating of a acrylic acid polymer or copolymer, or a coating
of a methacrylic acid polymer or copolymer on a glass or polymeric
support. Copolymers include, but are not limited to, copolymers
that contain polyethylene and polyacrylic acid or polymethacrylic
acid. Suitable polymeric support materials include polyesters,
polyimides, and the like.
A haloalkyl group can react, for example, with a substrate having a
tertiary amino group to form a quaternary ammonium-containing
attachment group. Suitable substrates having a tertiary amino group
include, but are not limited to, polydimethylaminostyrene or
polydimethylaminoethylmethacrylate.
Likewise, a tertiary amino group can react, for example, with a
substrate having a haloalkyl group to form a quaternary
ammonium-containing attachment group. Suitable substrates having a
haloalkyl group include, for example, coatings of a haloalkylsilane
on a support material. Support materials can include, but are not
limited to, glass and polymeric materials such as polyesters and
polyimides.
A primary aromatic amino or a secondary aromatic amino group can
react, for example, with a substrate having an isocyanate group to
form a oxycarbonylimino-containing attachment group. Suitable
substrates having isocyanate groups include, but are not limited
to, a coating of a 2-isocyanatoethylmethacrylate polymer or
copolymer on a glass or polymeric support. Suitable polymeric
supports include polyesters, polyimides, and the like.
A primary aromatic amino or a secondary aromatic amino group can
also react with a substrate containing a carboxy or halocarbonyl
group to form a carbonylimino-containing attachment group. Suitable
substrates include, but are not limited to, acrylic or methacrylic
acid polymeric coatings on a support material. The support material
can be, for example, glass or a polymeric material such as
polyesters or polyimides. Other suitable substrates include
copolymers of polyethylene and polymethacrylic acid or polyacrylic
acid.
A disulfide or an alkyl disulfide group can react, for example,
with a metal surface to form a metal sulfide-containing attachment
group. Suitable metals include, but are not limited to gold,
platinum, palladium, nickel, copper, and chromium. The substrate
can also be an alloy such an indium tin oxide or a dielectric
material.
A benzotriazolyl can react, for example, with a substrate having a
metal or metal oxide surface. Suitable metals or metal oxides
include, for example, silver, aluminum, copper, chromium, iron,
cobalt, nickel, zinc, and the like. The metals or metal oxides can
include alloys such as stainless steel, indium tin oxide, and the
like.
A phosphonitrilic can react with a substrate having amino
functionality associated with the surface of the substrate. Glass
surfaces and diamond-like glass surfaces treated with an
aminosilane are suitable for reacting with and attaching to a
phosphonitrilic moiety. In a such a DLG substrate, the amino
functionality provides a complementary functional group "G" capable
of reacting with an X group (e.g., chlorine) on the phosphonitrilic
moiety by nucleophilic attack. In the resulting system, the X group
is replaced by the amino functionality, thus tethering the
phosphonitrilic moiety to the substrate.
A phosphono, phosphoroamido, or phosphato can react, for example,
with a substrate having a metal or metal oxide surface. Suitable
metals or metal oxides include, for example, silver, aluminum,
copper, chromium, iron, cobalt, nickel, zinc, and the like. The
metals or metal oxides can include alloys such as stainless steel,
indium tin oxide, and the like.
An ethylenically unsaturated group can react, for example, with a
substrate having an alkyl group substituted with a mercapto group.
The reaction forms a heteroalkylene-containing attachment group.
Suitable substrates include, for example, mercapto-substituted
alkyl esters of polyacrylates or polymethacrylates.
An ethylenically unsaturated group can also react with a substrate
having a silicon surface, such as a silicon substrate formed using
a chemical vapor deposition process. Such silicon surfaces can
contain --SiH groups that can react with the ethylenically
unsaturated group in the presence of a platinum catalyst to form an
attachment group with silicon bonded to an alkylene group.
Additionally, an ethylenically unsaturated group can react with a
substrate having a carbon-carbon double bond to form an
alkylene-containing attachment group. Such substrates include, for
example, polymers derived from butadiene.
Articles according to the invention typically include a substrate
and a substrate-attached tethering group that includes a reaction
product of a complementary substrate-functional group on a surface
of the substrate with a phosphonitrilic compound of Formula I
(e.g., PNC) where the substrate-attached functional group is a
group capable of reacting with X to form an ionic bond, a covalent
bond, or combinations thereof. In some embodiments, a single
complementary substrate functional group may react to form more
than one bond with a single phosphonitrilic compound. Unreacted
groups on the substrate-attached functional group (e.g., unreacted
chloride) are available for further reaction with
nucleophile-containing materials.
More than one phosphonitrilic tethering group is typically attached
to the substrate if there are more than one reactive group on the
substrate. Further, the substrate can have excess reactive groups
on the surface of the substrate that have not reacted with a
phosphonitrilic tethering compound.
Groups on a substrate that are capable of reacting with the
phosphonitrilic tethering compound include, but are not limited to,
hydroxy, mercapto, primary aromatic amino group, secondary aromatic
amino group, secondary aliphatic amino group, aminosilane, azido,
carboxy, carbonyloxycarbonyl, isocyanate, halocarbonyl,
halocarbonyloxy, silanol, and nitrile.
The attachment of tethering compounds to the surface of a substrate
(i.e., formation) can be detected using techniques such as, for
example, contact angle measurements of a liquid on the substrate
before and after attachment of a phosphonitrilic tethering compound
(e.g., the contact angle can change upon attachment of a tethering
group to the surface of a substrate), ellipsometry (e.g., the
thickness of the attached layer can be measured), time-of-flight
mass spectroscopy (e.g., the surface concentration can change upon
attachment of a tethering group to a substrate), and Fourier
Transform Infrared Spectroscopy (e.g., the reflectance and
absorbance can change upon attachment of a tethering group to a
substrate).
In some embodiments of articles of the invention, a
halogen-containing moiety in the phosphonitrilic tethering group is
reacted with an amine-containing material resulting in the
immobilization of an amine-containing material to the substrate. In
some embodiments, the amine-containing materials are biomolecules
such as, for example, amino acid, peptide, DNA, RNA, protein,
enzyme, organelle, immunoglobin, or fragments thereof. In other
embodiments, the amine-containing material is a non-biological
amine such as an amine-containing analyte. The presence of the
immobilized amine can be determined, for example, using mass
spectroscopy, contact angle measurement, infrared spectroscopy, and
ellipsometry. Additionally, various immunoassays and optical
microscopic techniques can be used if the amine-containing material
is a biologically active material.
Other materials can be bound to the amine-containing material. For
example, a complementary RNA or DNA fragment can hybridize with an
immobilized RNA or DNA fragment. In another example, an antigen can
bind to an immobilized antibody or an antibody can bind to an
immobilized antigen. In a more specific example, a bacterium such
as Staphylococcus aureus can bind to an immobilized
biomolecule.
Another aspect of the invention provides methods for immobilizing a
nucleophile-containing material to a substrate. The method involves
preparing a substrate-attached tethering group by reacting a
complementary functional group on the surface of the substrate with
a phosphonitrilic moiety (e.g., reacting at least one of the
reactive groups X in compounds of Formula I); and reacting at least
one reactive group of the phosphonitrilic moiety (e.g., one or more
of the remaining reactive groups X of Formula I) with a
nucleophile-containing material to form a phosphonitrilic connector
group between the substrate and the nucleophile-containing
material. In one embodiment, the nucleophile-containing material is
an amine-containing material and the method of immobilizing the
amine-containing material is represented in Reaction Scheme A:
##STR00005## where U.sup.1 is the attachment group formed by
reacting one X group in a compound of Formula I with a
complementary functional group G on the surface of the substrate; T
is the remainder of the amine-containing material (e.g., the group
T represents all of the amine-containing material exclusive of the
amine group). H.sub.2N-T is any suitable amine-containing material.
In some embodiments, H.sub.2N-T is a biomolecule.
Variations of the foregoing Reaction Scheme A are also within the
scope of the invention. In embodiments where monofunctional
moieties are bonded to a phosphonitrilic moiety, methods involve
preparing a substrate-attached tethering group by reacting a
complementary functional group on the surface of the substrate with
the phosphonitrilic group (e.g., at least one of the reactive
groups X in compounds of Formula I), and reacting the
phosphonitrilic group (e.g., another of the remaining reactive
groups X of Formula I) with one or more monofunctional moieties to
form a tethering group that includes a phosphonitrilic moiety
bonded to a substrate with a monofunctional moiety also bonded to
the phosphonitrilic moiety. A nucleophile-containing material may
be bonded to the phosphonitrilic moiety to tether the nucleophile
containing material to the substrate.
In embodiments having a difunctional moiety, the difunctional
moiety is bonded to a first phosphonitrilic moiety that is tethered
to the surface of a substrate. The difunctional moiety may also be
bonded to a second phosphonitrilic moiety, and the second
phosphonitrilic moiety may be bonded to a nucleophile-containing
material to tether the nucleophile-containing material to the
substrate. In embodiments comprising multifunctional moieties, the
multifunctional moiety may be bonded to a first phosphonitrilic
moiety that is tethered to the surface of a substrate and the
multifunctional moiety may also be bonded to a second, third, or
other additional phosphonitrilic moieties. In turn, reactive groups
on the first, second, third or other phosphonitrilic moiety may
react with and bond to a nucleophile-containing material to tether
the nucleophile-containing material to the substrate. Additionally,
multifunctional moieties may react with multiple reactive groups on
single phosphonitrilic groups.
Accordingly, a method of immobilizing a nucleophile-containing
material to a substrate is provided, the method involving:
Providing a phosphonitrilic tethering compound (e.g., a compound
according to Formula I);
Providing a substrate having a complementary functional group
capable of reacting with a phosphonitrilic tethering compound;
Preparing a substrate-attached phosphonitrilic tethering group by
reacting the phosphonitrilic tethering compound with the
complementary functional group on the substrate resulting in an
ionic bond, covalent bond, or combinations thereof, and
Reacting the substrate-attached phosphonitrilic tethering group
with a nucleophile-containing material to immobilize the
nucleophile-containing material.
The compounds of the invention can be used, for example, for
immobilizing nucleophile-containing material such as an
amine-containing material. In some embodiments, the
amine-containing material is an amine-containing analyte. In other
embodiments, the amine-containing materials are biomolecules such
as, for example, amino acids, peptides, DNA, RNA, protein, enzymes,
organelles, immunoglobins, or fragments thereof. Immobilized
biological amine-containing materials can be useful in the medical
diagnosis of a disease or of a genetic defect. The immobilized
amine-containing materials can also be used for biological
separations or for detection of the presence of various
biomolecules. Additionally, the immobilized amine-containing
materials can be used in bioreactors or as biocatalysts to prepare
other materials. The substrate-attached tethering groups can be
used to detect amine-containing analytes.
Biological amine-containing materials often can remain active after
attachment to the substrate so that an immobilized antibody can
bind with antigen or an immobilized antigen can bind to an
antibody. An amine-containing material can bind to a bacterium. In
a more specific example, the immobilized amine-containing material
can bind to a Staphylococcus aureus bacterium (e.g., the
immobilized amine-containing material can be a biomolecule that has
a portion that can specifically bind to the bacterium).
The embodiments of the invention are further described in the
following non-limiting Examples.
EXAMPLES
Example 1
A functionalized porous membrane coated with diamond-like glass
(DLG) was prepared. A 5 cm.sup.2 high density polyethylene
thermally induced phase separation (HDPE TIPS) membrane (obtained
from 3M Company, St. Paul, Minn.) with a pore size of about 0.09 um
and having a thickness of about 23 micrometers was diamond like
glass (DLG) coated, using a plasma process as described in EP 1 266
045 B1 (David et al) to extend the DLG coating into the pores of
the TIPS membrane. The DLG-coated TIPS membrane was placed in 50 ml
of ethanol containing 2% by volume 3-amino propyl triethoxy silane
(Sigma-Aldrich, St. Louis, Mo.), 1 ml water and few drops of 0.1N
acetic acid. After 10 minutes in this solution the membrane was
removed and washed with ethanol and dried.
A PNC trimer was tethered on the functionalized membrane by placing
the membrane in 20 ml of a toluene solution containing 0.2 g of PNC
(Sigma Aldrich, St. Louis, Mo.) which was purified by sublimation.
The amino group of the aminosilane was reacted with the phosphazene
ring by displacing a chlorine, leaving the remaining chlorines
available for attachment to a biologically active molecule such as
a protein molecule. The membrane was placed in a solution of
glucose oxidase containing 10 mg glucose oxidase in PBS buffer
solution for 3 hours. The membrane was removed and washed with
water and buffer solution followed by washes with sodium
dodecylsulfate to remove any ionically bound proteins.
Bicinchonic acid analysis (BCA) was performed on the membrane from
Example 1 using a commercial protein assay kit and procedure
(Pierce Chemicals, Rockford, Ill.) to determine the total amount of
protein that had been immobilized on the surface. The amount of
total protein immobilized in a 1 cm.sup.2 TIPS porous membrane was
determined to be 212 .mu.g/1.5 mg of membrane.
A glucose oxidase assay was performed to determine the amount of
enzyme that was active in the membrane. The assay utilized a
glucose oxidase assay kit using a procedure obtained from
Sigma-Aldrich. The amount of enzyme active was initially determined
to be 25.5 .mu.g/1.5 mg of membrane. After five (5) days, the
amount of enzyme active was 23.3 .mu.g/1.5 mg of membrane.
An experiment was conducted to demonstrate that the enzyme activity
is attributable to the covalent attachment of the enzyme to
tethering groups on the surface of the membrane and not from the
unattached enzyme in solution. A 1 cm.sup.2 substrate, prepared as
described above, was placed in the glucose oxidase assay solution
for 30 seconds and the absorbance at 450 nm was measured. The
membrane was then removed from the solution for about 30 seconds
and the absorbance was checked again. No increase in absorbance was
noted for the membrane after it was removed from solution, thus
indicating a lack of free floating enzyme. The membrane was placed
back into the solution to allow further reaction to take place
between the enzymes in solution and the tethering groups on the
surface of the substrate. Additional absorbance measurements were
collected for 60 minutes, and the data is summarized in Table
1.
TABLE-US-00001 TABLE 1 Absorbance at 450 nm Time Absorbance (min)
(nm) 0 0 10 0.31979 20 0.36018 30 0.47089 40 0.49865 50 0.62823 60
0.64694
Example 2
Glass slides were treated with DLG using the following conditions.
Each glass slide was etched in oxygen plasma for 10 seconds and
exposed to a mixture of tetramethylsilane and oxygen plasma for 20
seconds followed by oxygen plasma for another 10 seconds. The DLG
coated glass slides were then placed in a 1% solution of
3-aminopropyltriethoxy silane in ethanol for 10 minutes.
Thereafter, the glass slides were removed and washed with ethanol
and dried under a nitrogen flow. The dried glass slides were
reacted with phosphoric chloride in toluene (Sigma Aldrich, St.
Louis, Mo.). The reaction time was varied from several minutes up
to one hour. Contact angle measurements were taken to monitor and
confirm attachment of the PNC to the aminopropyltriethoxy silane
attached to the DLG substrate. The amine has a low contact angle of
20 degrees, which on reaction with PNC increases to 45 degrees and
which stabilized in about 10 minutes. Contact angle data for the
attachment of the PNC is provided in Table 2.
TABLE-US-00002 TABLE 2 Time (min) Contact angle 0 19.3 1 22.3 5
45.3 20 44.3 30 47.3 60 46.6
The sample with a 10 minute reaction time was further reacted with
lysine by exposing the sample to a 1 mM solution of lysine (Sigma
Aldrich). The reaction of the amino group of lysine to the PNC
resulted in a decreased contact angle which stabilized within about
10 minutes following contact between the DLG coated slide and the
lysine. Contact angle data is set forth in Table 3.
TABLE-US-00003 TABLE 3 Time (min) Contact angle 0 55.2 1 27 5 15.5
20 15.3 30 19.3 60 15.7
Example 3
An approximately 20 cm by 30 cm polyimide film (obtained from E. I.
du Pont de Nemours & Co., Wilmington, Del. under the trade
designation "KAPTON E") was first coated with diamond-like carbon
(DLC) followed by diamond-like glass (DLG). The polyimide film was
affixed to the powered electrode of a Model 2480 parallel-plate
capacitively coupled reactive ion etcher (Plasma Therm, St.
Petersburg, Fla.) using 3M 811 adhesive tape (3M Company, St. Paul,
Minn.). DLC was deposited onto the polyimide membrane using an
acetylene plasma. The ion etcher chamber was closed and the chamber
was pumped to a pressure of 0.67 Pa (0.005 Torr). Oxygen gas was
introduced into the chamber at a flow rate of 500 standard cm.sup.3
per minute, and the pressure of the chamber was maintained at 6.7
Pa (0.050 Torr). Plasma was ignited and was sustained at a power of
2000 W for 15 seconds. The oxygen gas flow was then terminated and
the chamber was allowed to pump to a pressure of 0.67 Pa (0.005
Torr). Acetylene gas was introduced into the chamber at a flow rate
of 200 standard cm.sup.3 per minute, and the pressure of the
chamber was maintained at 2 Pa (0.015 Torr). Plasma was ignited and
was sustained at a power of 1600 W for 10 seconds. The flow of
acetylene gas was then terminated and the chamber was allowed to
pump to a pressure of 0.67 Pa (0.005 Torr).
Diamond-like glass (DLG) was thereafter deposited onto the
DLC/polyimide substrate using a tetramethylsilane plasma by first
introducing oxygen gas into the chamber at a flow rate of 500
standard cm.sup.3 per minute. The pressure of the chamber was
maintained at 20 Pa (0.15 Torr). Plasma was ignited and was
sustained at a power of 300W for 10 seconds. With the oxygen flow
rate maintained at 500 standard cm.sup.3 per minute,
tetramethylsilane gas was introduced into the chamber at a flow
rate of 150 standard cm.sup.3 per minute. The chamber pressure was
maintained at 20 Pa (0.15 Torr). Plasma was ignited and was
sustained at a power of 300 W for 12 seconds. The flow of
tetramethylsilane gas was terminated. After a period of 1 minute,
with both the flow of oxygen gas and the chamber pressure of 20 Pa
(0.15 Torr) maintained, plasma was ignited and was sustained at a
power of 300W for 20 seconds. The flow of oxygen gas was then
terminated and the chamber pressure was allowed to pump to a
pressure of 0.67 Pa (0.005 Torr). The chamber was then opened to
the atmosphere and the polyimide/DLC/DLG substrate was repositioned
so that the DLG coating faced the electrode. The foregoing sequence
of plasma treatments was repeated to provide a substrate with
polyimide having DLC/DLG coatings on both sides.
Two test substrates, each measuring about 1 cm.sup.2, were cut from
the 20 cm.times.30 cm polyimide/DLC/DLG substrate prepared
according to the foregoing process. One of the substrates was
designated as a control. The other substrate was designated as an
experimental substrate, and the experimental substrate was further
treated with 3-amino propyl triethoxy silane (Sigma-Aldrich, St.
Louis, Mo.) and a PNC trimer as in Example 1. The control substrate
was not treated and remained free of silane as well as PNC.
Mouse IgG against human (mIgG) was immobilized onto the DLG surface
of the control substrate and the PNC treated surface of the
experimental substrate by placing each substrate in a sterile
culture tube and exposing the substrate to 1 ml of 100 mM CHES
2-{N-cyclohexylaminoethane} sulfonic acid buffer (commercially
obtained from Sigma, St. Louis, Mo. under the catalog number
C-2885), adjusted pH to 9, containing 50 .mu.g of mIgG
(commercially obtained from Jackson Immuno Research laboratories
Inc West Grove, Pa., under catalog #209-005-082). The
immobilization time for general assays was set to be two (2) hours
while placed in a shaker (IKA HS 260 basic) at 120 motions/min at
room temperature. The solution was removed from the culture tube by
Pasteur pipette and the thus treated control and experimental
substrates were washed three times with Phosphate buffered saline
(PBS) buffer containing 0.05% Tween 20. Both of the substrates were
again placed in sterile culture tubes and 1.5 ml of blocking
buffer, PBS buffer containing 2% non-fat milk powder, was added to
each of the culture tubes and allowed to react for one (1) hour
while on the shaker. The solution was removed from the tubes with a
Pasteur pipette and each of the substrates were again washed three
times with the forgoing wash buffer.
The mIgG antibody was reacted with biotin-conjugated human IgG
(hIgG-BT). The concentration of the hIgG-BT was 4 .mu.g/ml
(obtained from Jackson Immuno Research laboratories Inc West Grove,
Pa., under catalog, #009-060-003) in PBS buffer. A volume of 1 ml
of the solution was placed in the culture tube containing the
substrate and incubated for one (1) hour in the shaker, and the
substrates were then washed 3 times with buffer as previously
described. This reaction was followed by reaction with streptavidin
horse radish peroxide (SA-HRP), a detection enzyme that
specifically binds to biotin. A volume of 1 ml of 0.5 .mu.g/ml of
SA-HRP (commercially obtained from Jackson Immuno Research
laboratories Inc West Grove, Pa., under Catalog #023-060-021 in pH
7.4 buffer) was added to the culture tube and allowed to react for
30 minutes on a shaker. The samples were again washed 3 times with
wash buffer, and a 1 ml volume of the coloring agent
2,2-azino-di(3-ethylbenzthiazoline) sulfonic acid (ABTS) at a
concentration of 0.3 mg/ml was added to the culture tube to promote
an enzymatic color change that could be measured at 405 nm on a
spectrometer. After a 5 minute exposure to the ABTS, 1 ml of 1%
Sodium Dodecyl sulfate (SDS) solution was added to stop the
reaction.
Absorbance was measured for both the treated control substrate and
for the treated experimental substrate using a UV-Vis
spectrophotometer at 405 nm. Absorbance for the sample on the
control was: 0.1. Absorbance for the sample on the experimental
substrate was: 0.2
Example 4
1 cm.sup.2 substrate (polyimide/DLC/DLG) samples were prepared and
functionalized with silane and PNC as described in Example 3.
Polyimide/DLC/DLG substrate with no silane or PNC treatment were
used as `control` substrates. Rabbit IgG specific to Staphylococcus
Aureus (commercially obtained from Accurate Chemical and
Scientific, Westbury, New York) in a 4.52 mg/ml solution was
immobilized on the surface of the substrates, including the control
substrate. The substrates were then fixed (by taping) on a glass
plate and 50 .mu.l of PBS buffer containing Staphylococcus aureus
at a concentration of 5.times.10.sup.8 cfu/ml was added by
pipetting the solution and allowing it to stand for approximately
30 minutes. The samples were washed and then stained by exposing
each of the samples to acridine orange for 10 minutes. The acridine
orange (obtained from Molecular probes under the designation A3568)
was diluted with distilled water from a concentration of 10 mg/ml
to 0.1 mg/ml prior to use.
Each of the thus stained substrates were viewed through a Olympus
Model FV-300 confocal microscope (Leeds Precision, Inc, of
Minnesota). The PNC functionalized substrates were observed to
include a higher level of stained bacteria when compared with the
non-functionalized control substrates, indicating the PNC
functionalized substrates bound more of the S. aureus bacteria
compared to the non-functionalized control samples.
* * * * *